Amphotericin B products are used to treat a variety of fungal infections, including systemic fungal infections.
Currently, polyenes are a major drug of choice for serious, disseminated fungal infections. Yet, the usefulness of polyenes is often limited by a lack of efficacy and by significant toxicity. These considerations, together with the predicted increased incidence of disseminated fungal infections, provide a strong impetus for the development of new antifungal therapies or improvement of existing ones. In regard to the latter approach, multiple polyenes are produced by Streptomyces noduses and could be useful as lead compounds. The present inventors observed antimycotic activity by many of these polyene compounds, but their cellular toxicity appeared high relative to the polyene compound known as amphotericin B (AmB). AmB is commercially available in a variety of formulations, however the active pharmaceutical ingredient called AmB contains other polyene compounds in addition to the compound known as AmB. The present inventors discovered that some of these non-AmB polyenes and perhaps other contaminants present in commercial AmB formulations were responsible for enhanced adverse effects of the drug (J. D. Cleary, R. Kramer, E. Swiatlo, and S. W. Chapman, Abstr. 45th Intersci. Conf. Antimicrob. Agents Chemother., abstr. F498, 2005).
Although the present invention encompasses many pharmaceutical formulations, AmB is used primarily in intravenous formulations in the treatment of severe fungal infections. However, its usefulness is compromised by a high incidence of adverse effects [flu-like symptoms (fever, chills, myalgias), capillary leak syndrome (hypotension, decreased organ perfusion), pulmonary congestion, changes in mental status (lethargy, confusion, agitation), renal dysfunction with secondary hypokalemia, hypomagnesemia and anemia, and liver dysfunction]. These adverse reactions are observed in up to seventy percent of treated patients. The mechanisms responsible for these reactions are, to date, not entirely known.
Yet, today (and for the past 40 years) AmB remains the best or only alternative for critically ill patients. Excerpts from the AmB boxed warning include the following:
Contraindications                This product is contraindicated in those patients who have shown hypersensitivity to amphotericin B or any other component in the formulation unless, in the opinion of the physician, the condition requiring treatment is life-threatening and amenable only to amphotericin B therapy.        
Warnings                Amphotericin B is frequently the only effective treatment available for potentially life-threatening fungal disease. In each case, its possible life-saving benefit must be balanced against its untoward and dangerous side effects.        
Precautions                Amphotericin B should be administered intravenously under close clinical observation by medically trained personnel. It should be reserved for treatment of patients with progressive, potentially life-threatening fungal infections due to susceptible organisms (see INDICATIONS AND USAGE).        Acute reactions including fever, shaking chills, hypotension, anorexia, nausea, vomiting, headache, and tachypnea are common 1 to 3 hours after starting an intravenous infusion. These reactions are usually more severe with the first few doses of amphotericin B and usually diminish with subsequent doses.        Rapid intravenous infusion has been associated with hypotension, hypokalemia, arrhythmias, and shock and should, therefore, be avoided (see DOSAGE AND ADMINISTRATION).        Amphotericin B should be used with care in patients with reduced renal function; frequent monitoring of renal function is recommended (see PRECAUTIONS, Laboratory Tests and ADVERSE REACTIONS). In some patients hydration and sodium repletion prior to amphotericin B administration may reduce the risk of developing nephrotoxicity. Supplemental alkali medication may decrease renal tubular acidosis complications.        Since acute pulmonary reactions have been reported in patients given amphotericin B during or shortly after leukocyte transfusions, it is advisable to temporally separate these infusions as far as possible and to monitor pulmonary function (see PRECAUTIONS, Drug Interactions).        Leukoencephalopathy has been reported following use of amphotericin B. Literature reports have suggested that total body irradiation may be a predisposition.        
Whenever medication is interrupted for a period longer than seven days, therapy should be resumed by starting with the lowest dosage level, e.g., 0.25 mg/kg of body weight, and increased gradually as outlined under DOSAGE AND ADMINISTRATION.
As can be seen from the label information, AmB is used to treat a variety of life threatening infections despite very serious and dangerous side effects.
The United States Pharmacopeia (USP) has established quality standards for AmB used in pharmaceutical formulations. The USP allows AmB used as an active ingredient in pharmaceutical formulations to contain up to 25% impurities, including multiple polyene components. Unlike most active pharmaceutical ingredients, which have impurity levels less than 1%, AmB is not a single compound with very low impurity levels. See, for example, J. Antimicro Chemother 2007 60(6): 1331-1340. To comply with the USP, AmB formulations must have a potency of not less than 750 micrograms in 1 mg of material, a purity of 75%. Despite the fact that AmB, USP may contain only 75% of the polyene compound known as AmB, the entire pharmaceutical formulation is labeled “amphotericin B” as though it were all the active ingredient.
By discovering the source of the side effects, the present inventors have met a long-felt need in allowing a safer product. This was previously not known, and is surprising when compared to the many prior attempts to formulate a safer AmB product by adding other components to AmB containing formulations in hopes of reducing toxic effects or by using other drug products concurrently to reduce some of the side effects. Researchers in the field did not understand the underlying cause of the side effects despite over 40 years of dealing with significant adverse effects caused by the therapeutic use of AmB formulations. Thus, even though there were prior attempts to “purify” AmB formulations, there was no motivation to use the methods of the present invention, and there was no motivation to remove non-amphotericin B polyenes from the formulation. As stated above, it was the present inventors that discovered that the non-amphotericin B polyenes should be removed from AmB.
Through molecular biologic techniques, the present inventors have identified inflammatory cytokine genes that are up-regulated (increased in cells) after exposure to AmB. The genes include interleukin-1, a potent inflammatory cytokine. The adverse effects associated with stimulation of interleukin-1 are discussed below.
Proposed mechanisms of AmB-induced “flu like syndrome” include the expression of interleukin-1 (IL-1), tumor necrosis factor (TNF) or prostaglandins by mononuclear cells which then alter the hypothalamic set point inducing fever and chills. Administration of endotoxin causes similar reactions AmB exposure to mononuclear cells induces unique morphological changes and dramatically altered protein expression. Some host cell proteins have been reported to be inducible by AmB, such as TNF-α and IL-1β. The present inventors have demonstrated that this protein expression is not associated with release of preformed protein; protein release is associated with up-regulation of a gene or derepression of an inhibitory gene.
Currently, pharmacologic agents used to prevent AmB adverse reactions only address small aspects of the problem. Hydrocortisone is used to prevent the flu-like syndrome and hypotension. Acetaminophen is also used to prevent the flu-like syndrome. Fluids administered parenterally are used to prevent renal dysfunction. Also, lipid products have been developed to decrease the toxicity of AmB. These products encapsulate AmB in an attempt to reduce side effects. However, they are not totally successful.
What is needed, then, is an AmB active pharmaceutical ingredient that can be used in a variety of formulations without the high incidence of side effects.